Demonstration of receptors for parathyroid hormone on cultured rabbit costal chondrocytes

Parathyroid hormone (PTH) receptors on cultured rabbit costal chondrocytes were demonstrated using HPLC-purified, radioiodinated [Nle8,-Nle18, Tyr34] bovine PTH-(1-34)amide. PTH binding was found to be specific for PTH agonists and antagonists and dependent on the time and temperature of incubation. Both growth cartilage (GC) cells and resting cartilage (RC) cells were shown to have a single class of saturable, high affinity PTH binding sites with a dissociation constant of 0.6-0.7 nM. However, the numbers of receptors per cell were approximately 49,000 on GC cells and 19,000 on RC cells. After crosslinking the receptors on these cells with the radioligand, one, major 125I-labeled band of 76 kDa was separated by SDS-PAGE.     

Induction of spermidine/spermine N1-acetyltransferase by parathyroid hormone in rabbit costal chondrocytes in culture

Parathyroid hormone (PTH) increased the activity of spermidine/spermine N1-acetyltransferase, a rate-limiting enzyme of polyamine biodegradation, in rabbit costal chondrocytes in culture. The enzyme activity increased in a dose-dependent manner after addition of PTH to the culture, reaching a maximum at 8 h. The increase in the enzyme activity was abolished by cycloheximide or actinomycin D. Dibutyryl cyclic AMP also induced the acetyltransferase to some extent. These results suggest that the induction of spermidine/spermine N1-acetyltransferase by PTH may play some significant role in the expression of the differentiated phenotype of chondrocytes.     

Effect of exogenous extracellular matrices on proteoglycan synthesis by cultured rabbit costal chondrocytes

We examined the effect of an extracellular matrix (ECM), produced by either bovine corneal endothelial (BCE) cells or mouse PF HR-9 teratocarcinoma cells, on the ability of rabbit costal chondrocytes to re-express their phenotype once confluent. Rabbit chondrocytes seeded at low densities and grown on plastic tissue culture dishes produced a heterogeneous cell population composed of both overtly differentiated and poorly differentiated chondrocytes, as well as fibroblastic cells. On the other hand, cultures grown on BCE-ECM- or HR-9-ECM-coated dishes reorganized into a homogeneous cartilage-like tissue composed of round cells surrounded by a refractile matrix that stained intensely with alcian green. The cell ultrastructure and that of their pericellular matrix were similar to those seen in vivo. The differentiation of chondrocyte cultures grown on the ECMs vs. plastic was reflected by a two- to three-fold increase in the maximal rate of incorporation of [35S]sulfate and [3H]glucosamine into proteoglycans. Furthermore, the ratio of 35S-labeled proteoglycans incorporated in the cell layer vs. those released into the medium was 1.5-2.5-fold higher when cultures were grown on the ECMs than on plastic. This suggests that the ECMs stimulate the incorporation of newly synthesized proteoglycans into a cartilaginous matrix. Since chondrocyte cultures grown on BCE-ECM or HR-9-ECM give rise to a homogeneous cartilage-like tissue even when seeded at low cell densities, they provide a model for the study of cell-substrate interactions that are responsible for the maintenance of the differentiated phenotype of chondrocytes.     

Differential effects of parathyroid hormone and somatomedin-like growth factors on the sizes of proteoglycan monomers and their synthesis in rabbit costal chondrocytes in culture

In the proteoglycans extracted from rabbit costal chondrocytes in culture, two populations of proteoglycans were distinguished by density gradient centrifugation under dissociative conditions. The major component was the faster sedimenting population (proteoglycan I), the putative 'cartilage-specific' proteoglycans, and the minor component was the slower sedimenting population (proteoglycan II). The monomeric size of proteoglycan I was closely related to the differentiation-state of chondrocytes and was a good marker of the differentiated chondrocytes. Treatment of the cultures with parathyroid hormone (PTH) induced an increase in the monomeric size of proteoglycan I. This increase was ascribed to an increase in the molecular size of the glycosaminoglycan chain in proteoglycan I. On the other hand, somatomedin-like growth factors, such as multiplication-stimulating activity (MSA) and cartilage-derived factor (CDF), did not affect the size of proteoglycan I, while they markedly stimulated the synthesis of proteoglycan I. In contrast, treatment with nonsomatomedin growth factors, such as fibroblast growth factor (FGF) and epidermal growth factor (EGF), resulted in not only a decrease in glycosaminoglycan synthesis but also a slight decrease in size of proteoglycan I. However, synthesis and size of proteoglycan II were little affected by these agents. Thus, the present study clearly shows that PTH and somatomedin-like growth factors have differential functions in bringing about the expression of the differentiated phenotype of chondrocytes: PTH influences chain elongation and termination of glycosaminoglycans in proteoglycan I, while somatomedin-like growth factors affect primarily the synthesis and secretion of proteoglycan I.     

Effects of static magnetic field and pulsed electromagnetic field on viability of human chondrocytes in vitro

Effects of electromagnetic fields (EMFs) on human cell lines were described in numerous studies, but still many questions remain unanswered. Our experiment was designed with the aim of studying the effects of EMFs on the metabolic activity of chondrocytes in vitro. Human chondrocyte in vitro cultures, cultured in medium supplemented with 20 % fetal calf serum, were exposed to static magnetic field (SMF) (intensity of 0.6 T) and pulsed electromagnetic fields (PEMF) (21.2 MHz period of 15 ms, burst duration of 2 ms, amplification 3 dBm (0.1 V) and maximum output of 250 W) continually for 72 h. After the exposure, viability was determined using the MTT test and compared with a non-exposed control culture. As compared to the control sample the exposure to SMF resulted in a statistically significant increase (p 0.001) in viability. However, the increase of viability after PEMF exposure was not significant. This could be due to the frequency dependent effect on human cells. The experiments demonstrated that magnetic fields, using the above parameters, have a positive effect on the viability of human chondrocytes cultured in vitro.     

The metabolism of hyaluronan in cultured rabbit growth plate chondrocytes during differentiation

Hyaluronan (HA) is one of the major extracellular matrix components in cartilage. In addition to the biomechanical functions, HA has various important roles in the differentiation of chondrocytes. The purpose of this study was to clarify the nature of HA synthesis during chondrocyte differentiation. Growth plate chondrocytes were isolated from rabbit ribs and cultured in chondrocyte differentiation medium. The amount of HA and HA synthase (HAS) mRNA levels were analyzed for each stage of chondrocyte differentiation by means of high-performance liquid chromatography (HPLC) and real-time PCR, respectively. The distribution of HA in cultured chondrocytes was observed by histochemical staining. The amount of HA, ranging widely in size, was increased substantially during the hypertrophic stage. The expression levels of HAS2 and HAS3 mRNAs were low during the matrix-forming stage. HAS2 mRNA level was substantially enhanced at the pre-hypertrophic stage, whereas HAS3 mRNA level exhibited a slight increase. HAS1 mRNA was not detected. The intensity of HA staining was high around the hypertrophic chondrocytes. These results suggest that HA metabolism in chondrocyte differentiation is regulated by the selective expression of HASs, and HAS2 and the related large size-HA may have a certain association with the hypertrophic changes of chondrocytes.     

In vitro exposure of human chondrocytes to pulsed electromagnetic fields

The effect of pulsed electromagnetic fields (PEMFs) on the proliferation and survival of matrix-induced autologous chondrocyte implantation (MACI)-derived cells was studied to ascertain the healing potential of PEMFs. MACI-derived cells were taken from cartilage biopsies 6 months after surgery and cultured. No dedifferentiation towards the fibro- blastic phenotype occurred, indicating the success of the surgical implantation. The MACI-derived cultured chondrocytes were exposed to 12 h/day (short term) or 4 h/day (long term) PEMFs exposure (magnetic field intensity, 2 mT; frequency, 75 Hz) and proliferation rate determined by flow cytometric analysis. The PEMFs exposure elicited a significant increase of cell number in the SG2M cell cycle phase. Moreover, cells isolated from MACI scaffolds showed the presence of collagen type II, a typical marker of chondrocyte functionality. The results show that MACI membranes represent an optimal bioengineering device to support chondrocyte growth and proliferation in surgical implants. The surgical implant of MACI combined with physiotherapy is suggested as a promising approach for a faster and safer treatment of cartilage traumatic lesions.     

Stimulation by glucocorticoids of the differentiated phenotype of chondrocytes and the proliferation of rabbit costal chondrocytes in culture

Hydrocortisone stimulated glycosaminoglycan (GAG) synthesis, a characteristic of the cartilage phenotype, of rabbit costal chondrocytes in confluent quiescent culture, as judged by the incorporations of [35S]sulfate and [3H]glucosamine. Hydrocortisone also stimulated incorporation of [3H]serine into proteoglycan. The stimulation of GAG synthesis by hydrocortisone was dose-dependent and maximal at a physiological concentration of 10(-7) M. Hydrocortisone also stimulated GAG synthesis in cultures in the log-phase of growth. In this case, its maximal effect was observed at a concentration of 10(-6) M. The magnitude of the increase of GAG synthesis in response to hydrocortisone was larger in confluent culture than in log-phase cultures. Hydrocortisone stimulated DNA synthesis dose-dependently, and its effect was observable at a physiological concentration. However, no stimulation of DNA synthesis by hydrocortisone was observed in serum-free medium, in contrast to that of GAG synthesis. Hydrocortisone also increased protein synthesis and the cell number. Dexamethasone also stimulated the syntheses of both GAG and DNA. These results show that glucocorticoids stimulated both the differentiated phenotype of chondrocytes and the proliferation of rabbit costal chondrocytes in culture. Moreover, the effect of glucocorticoids was primarily on the differentiated phenotype of chondrocytes and its effect on proliferation was permissive.     

Effects of pulsed electromagnetic field on growth and differentiation of embryonal carcinoma cells

A murine embryonal carcinoma cell line (F9) was used to examine the effect of a pulsed electromagnetic field on the growth and differentiation of malignant cells. The cells can be induced to differentiate into parietal endodermal cells by treatment with retinoic acid. The pulsed electromagnetic field (1 Gauss and 10 Gauss) promoted the growth of embryonal carcinoma cells in both the presence and absence of retinoic acid. The pulsed electromagnetic field was also found to inhibit retinoic acid-induced differentiation, when the degree of differentiation was based on morphological criteria or on the production of plasminogen activator.     

Stimulation by glucocorticoid of the synthesis of cartilage-matrix proteoglycans produced by rabbit costal chondrocytes in vitro

The effect of glucocorticoids on sulfated proteoglycan synthesis by rabbit costal chondrocyte cultures exposed to serum-free conditions has been examined. Low density cultures of rabbit costal chondrocytes were maintained on dishes coated with extracellular matrix produced by bovine corneal endothelial cells and exposed to a 9:1 mixture (v/v) of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with transferrin, high density lipoproteins, fibroblast growth factor, and insulin (Medium A). Chondrocytes maintained in the presence of Medium A supplemented with 10(-7) M hydrocortisone reorganized, at confluence, into a homogeneous cartilage-like tissue composed of round cells surrounded by a refractile matrix in which abundant thin collagen fibrils characteristic of type II collagen were observed. The cell ultrastructure and fibrils of the pericellular matrix were similar to those seen in vivo. In contrast, cells maintained in the presence of Medium A alone, once they reached confluence, formed a fibroblastic multilayer and produced thick collagen bundles. The level of 35SO4(2-) incorporated into large cartilage-specific proteoglycans in glucocorticoid-supplemented cultures was 33-fold higher than that of glucocorticoid-free cultures. The level of 35SO4(2-) incorporated into small ubiquitous proteoglycans was only 4-fold higher than that of glucocorticoid-free cultures. On the other hand, the level of [3H]glucosamine incorporated into hyaluronate in glucocorticoid-supplemented cultures was 4.5-fold lower than that of glucocorticoid-free cultures. Within 24 h of their addition to confluent cultures, hydrocortisone or dexamethasone markedly stimulated proteoglycan synthesis. This effect was not mimicked by androgens, estrogens, progesterone, or an inactive form of glucocorticoids such as deoxycorticosterone. This suggests that glucocorticoids have a direct and specific stimulatory effect on cartilage-specific proteoglycan synthesis and are essential for the maintenance of this synthesis in low density chondrocyte cultures.     

Differential effects of parathyroid hormone responsive cultured human cells on biological activity of parathyroid hormone and parathyroid hormone inhibitory analogues

We have employed parathyroid hormone (PTH) responsive human cells cultured from dermis or giant cell tumors of bone (GT) to evaluate the biological properties of a newly developed in vivo PTH inhibitor, [Tyr34]bPTH-(7-34)-amide (PTH-Inh). Short periods of incubation of cells from dermis or GT with maximal stimulatory concentrations of PTH in the presence of increasing concentrations of PTH-Inh resulted in a dose-dependent inhibition of the adenosine cyclic 3',5'-phosphate (cAMP) response (Ki = 3 X 10(-7) M and 4.2 X 10(-7) M for GT and dermal cells, respectively). In both cell cultures, PTH-Inh alone did not increase cAMP levels, and in desensitization experiments, preincubation with PTH-Inh alone did not desensitize cells to PTH. Hence, the analogue displayed no agonist properties. Unexpectedly, when PTH-Inh was incubated with dermal cells in the presence of PTH, the PTH-Inh failed to block desensitization, suggesting a loss of biological effectiveness of the inhibitor. When medium containing PTH-Inh alone was removed from dermal cells and tested for inhibition of the acute PTH response in untreated cells, there was apparent loss of inhibitory efficacy (t1/2 = 20 h). In contrast, incubation of native PTH or bPTH-(1-34) with cells did not affect the biological activity of these ligands. Unlike the dermal cells, the PTH-Inh did block desensitization to PTH in GT, and there was no loss of inhibitor efficacy when medium containing PTH-Inh was incubated with GT (48 h) and then tested in untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extremely low frequency pulsed electromagnetic field designed for antinociception does not affect microvascular responsiveness to the vasodilator acetylcholine

A 225 µT, extremely low frequency, pulsed electromagnetic field (PEMF) that was designed for the induction of antinociception, was tested for its effectiveness to influence blood flow within the skeletal microvasculature of a male Sprague–Dawley rat model (n = 103). Acetylcholine (0.1, 1.0, or 10 mM) was used to perturb normal blood flow and to delineate differential effects of the PEMF, based on degree of vessel dilation. After both 30 and 60 min of PEMF exposure, we report no effects on peak perfusion response to acetylcholine (with only 0.2% of the group difference attributed to exposure). Spectral analysis of blood flow data was generated to obtain information related to myogenic activity (0.15–0.40 Hz), respiratory rate (0.4–2.0 Hz), and heart rate (2.0–7.0 Hz), including the peak frequency within each of the three frequency regions identified above, peak power, full width at half maximum (FWHM), and mean within band. No significant effects due to exposure were observed on myogenic activity of examined blood vessels, or on heart rate parameters. Anesthesia‐induced respiratory depression was, however, significantly reduced following PEMF exposure compared to shams (although exposure only accounted for 9.4% of the group difference). This set of data suggest that there are no significant acute physiological effects of 225 µT PEMF after 30 and 60 min of exposure on peak blood flow, heart rate, and myogenic activity, but perhaps a small attenuation effect on anesthetic‐induced respiratory depression. Bioelectromagnetics 31:64–76, 2010. © 2009 Wiley‐Liss, Inc.     

Cytoskeleton and differentiation: effects of cytochalasin B and colchicine on expression of the differentiated phenotype of rabbit costal chondrocytes in culture

Cytochalasin B changed the shape of cultured rabbit costal chondrocytes from polygonal to nearly spherical and stimulated glycosaminoglycan synthesis, which is a differentiated phenotype of chondrocytes, whereas colchicine changed them from polygonal to flattened and inhibited glycosaminoglycan synthesis. These morphological changes occurred parallel with the changes in glycosaminoglycan synthesis. Induction of ornithine decarboxylase by parathyroid hormone, which is a good marker of differentiated chondrocytes, was markedly potentiated in the spherical cells which had been pretreated with cytochalasin B, whereas pretreatment with colchicine inhibited the induction of the enzyme. Both cytochalasin B and colchicine inhibited DNA synthesis. The inhibitions were observed after the appearance of changes in the morphology of the cells and glycosaminoglycan synthesis. These findings suggest that intactness of microtubules and disruption of microfilaments are involved in regulating the expression of the differentiated phenotype of chondrocytes in culture.     

Biotinylated parathyroid hormone as a probe for the parathyroid hormone receptor. Structure-function analysis and detection of specific binding to cultured bone cells by flow cytometry

The synthetic bovine parathyroid hormone (PTH) analog (Nle8, Nle18, Tyr34) bovine PTH(1-34)amide (bPTH(1-34)amide) was reacted with biotinyl-epsilon aminocaproic acid-N-hydroxysuccinimide under conditions which yielded five isoforms which were fractionated by a combination of reversed phase and ion-exchange chromatography. These reaction products were analyzed by automated Edman degradation in a manner which allowed us to specify the location and number of biotin residues on picomole quantities of hormone. The ability of each of these isoforms to induce a rise in intracellular cAMP in the ROS 17/2.8 cell line allowed us to evaluate the effect on function of biotinylation at different residues. Derivatized PTH molecules which contained a single biotin at either lysine 13, lysine 26, or lysine 27 possessed full biological activity. However, bioactivity was significantly reduced when position 13 plus either lysine 26 or 27 were biotinylated. Biological activity was lost when all 3 lysine residues were biotinylated. Biotinylation of the alpha-NH2 group of alanine at the NH2 terminus also resulted in a total loss of activity. Hence, unlike the effect of altering the alanine at position 1, modification of a single lysine residue at positions 13, 26, and 27 has a less critical effect on biological activity of the molecule. However, biotinylation of all three lysines results in a biologically inert PTH derivative and suggests that changes in isoelectric point, hydrophobicity, or tertiary structure may strongly influence hormone function. A fully bioactive-mixture of isoforms was used to detect receptors on ROS 17/2.8 cells by flow cytometry using fluorescein isothiocyanate-avidin as a fluorescent indicator. Binding to cell surface receptors was saturable and could be inhibited by native bPTH(1-34) but not by transforming growth factor beta, calcitonin or insulin. Moreover, PTH receptors could also be detected on primary cultures of human bone cells and human fibroblasts.     

Abnormal limb regeneration in adult newts exposed to a pulsed electromagnetic field

This investigation examined two questions: 1) whether or not forelimb regeneration results in a faithful replacement of the distal skeletal pattern and 2) what effect exposure to a pulsed electromagnetic field (PEMF), the type reported to facilitate healing of human non-united bone fractures, would have on the process of limb regeneration. Of the native forelimbs, 98% (132 out 134) had a skeletal pattern that showed little difference with the only variation being the range of carpal bones (5-8). Following a 4-5 month postamputation period, the skeletal pattern of the normal regenerates was examined. While 72% (135 out of 188) of these forelimbs resembled the native group, 28% (53 out of 188) were abnormal. These abnormalities consisted of the loss of a digit, fused carpals, and long bone defects which occurred singly or in combination with one another. Exposure to a PEMF for the first 30 days postamputation, followed by a 3-4 month postamputation period, produced in addition to the normal (60%, 144 out of 240) and abnormal forelimbs (28%, 67 out of 240), a group of forelimbs with unique gross defects (12%, 28 out of 240). These defects, occurring singly or in combination, included the loss of two or more digits with associated loss of carpals, absence of the entire hand pattern, and abnormalities associated with the radius and ulna. We can offer no explanation for these observations.     

Protection against focal cerebral ischemia following exposure to a pulsed electromagnetic field

There is evidence that electromagnetic stimulation may accelerate the healing of tissue damage following ischemia. We undertook this study to investigate the effects of low frequency pulsed electromagnetic field (PEMF) exposure on cerebral injury in a rabbit model of transient focal ischemia (2 h occlusion followed by 4 h of reperfusion). PEMF exposure (280 V, 75 Hz, IGEA Stimulator) was initiated 10 min after the onset of ischemia and continued throughout reperfusion (six exposed, six controls). Magnetic resonance imaging (MRI) and histology were used to measure the degree of ischemic injury. Exposure to pulsed electromagnetic field attenuated cortical ischemic edema on MRI at the most anterior coronal level by 65% (P < 0.001). On histologic examination, PEMF exposure reduced ischemic neuronal damage in this same cortical area by 69% (P < 0.01) and by 43% (P < 0.05) in the striatum. Preliminary data suggest that exposure to a PEMF of short duration may have implications for the treatment of acute stroke. © 1994 Wiley-Liss, Inc.     

Conversion of proparathyroid hormone to parathyroid hormone by a particulate enzyme of the parathyroid gland.

The conversion of proparathyroid hormone (proparathormone) to parathyroid hormone (parathormone) by subcellular fractions of the bovine parathyroid has been investigated. The identification of the conversion product as parathormone was established by its elution postion during ion exchange chromatography and gel filtration, and by partial amino acid sequence analysis of its NH2-terminal region. Total homogenates and derived subcellular fractions (600 X g pellet, 5,000 X g pellet, 20,000 X g pellet, 190,000 X g pellet, and 190,000 X g supernatant) all catalyzed the conversion of exogenous [3H]- or [14C]prohormone. Over 60% of the converting activity was in the particulate fractions; the 190,000 X g particulate fraction contained the highest specific converting activity. The converting activity appeared to be an integral component of the membranes since it could only be partially removed by extraction with Triton X-100. The production of parathormone by the particulate converting enzyme increased with time and the concentration of enzyme protein. The optimum pH range was between 7 and 9, and the enzyme was inactive below pH 6. Conversion by the particulate enzyme was inhibited by benzamidine or chloroquine, but not by pancreatic trypsin inhibitor, indicating its dissimilarity to trypsin. When a mixture of [14C]proparathormone and [3H]parathormone was used as substrate, the particulate enzyme did not metabolize the hormone despite over 70% conversion of the prohormone to hormone and other peptides. There was a close correlation between the subcellular distribution of converting activity and that of newly formed parathormone found in the membrane fraction. These data suggest that the particulate converting activity is that concerned with the formation of parathormone in vivo.     

Up-regulation of per mRNA expression by parathyroid hormone through a protein kinase A-CREB-dependent mechanism in chondrocytes

In bone, clock genes are involved in the circadian oscillation of bone formation and extracellular matrix expression. However, to date little attention has been paid to circadian rhythm in association with expression of clock genes during chondrogenesis in cartilage. In this study, we investigated the functional expression of different clock genes by chondrocytes in the course of cartilage development. The mRNA expression of types I, II, and X collagens exhibited a 24-h rhythm with a peak at zeitgeber time 6, in addition to a 24-h rhythmicity of all the clock genes examined in mouse femurs in vivo. Marked expression of different clock genes was seen in both osteoblastic MC3T3-E1 and chondrogenic ATDC5 cells in vitro, whereas parathyroid hormone (PTH) transiently increased period 1 (per1) mRNA expression at 1 h in both cell lines. Similar increases were seen in the mRNA levels for both per1 and per2 in prehypertrophic chondrocytes in metatarsal organotypic cultures within 2 h of exposure to PTH. PTH significantly activated the mouse per1 (mper1) and mper2 promoters but not the mper3 promoter in a manner sensitive to both a protein kinase A inhibitor and deletion of the cAMP-responsive element sequence (CRE) in ATDC5 cells. In HEK293 cells, introduction of brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (bmal1)/clock enhanced mouse type II collagen first intron reporter activity without affecting promoter activity, with reduction effected by either per1 or per2. These results suggest that PTH directly stimulates mper expression through a protein kinase A-CRE-binding protein signaling pathway for subsequent regulation of bmal1/clock-dependent extracellular matrix expression in cartilage.